The present invention is in the field of recombinant mammalian viral vectors. More particularly, it concerns recombinant porcine adenovirus vectors for diagnostic and therapeutic purposes, such as vaccines and expression systems.
Adenoviruses are double-stranded DNA viruses that have been isolated from a wide variety of avian and mammalian species, including swine. While the majority of adenovirus infections in swine are subclinical, porcine adenovirus (PAV) infection has been associated with encephalitis, pneumonia, kidney lesions and diarrhea. Derbyshire (1992) In: xe2x80x9cDiseases of Swinexe2x80x9d (ed. Leman et al.), 7th edition, Iowa State University Press, Ames, Iowa. pp. 225-227. Thus, there is a need for vaccines that will provide protection against PAV infection.
In addition to their potential ability to provide protection against PAV infection, PAVs could also be used as viral vaccine vectors, if insertion capacity can be determined, and appropriate insertion sites can be defined and characterized. It has been shown that PAV is capable of stimulating both humoral response and a mucosal antibody responses in the intestine of infected piglets. Tuboly et al. (1993) Res. in Vet. Sci. 54:345-350. Thus, recombinant PAV vaccine vectors would be especially useful, as they would be likely to be capable of providing both systemic and mucosal immunity to antigens encoded by native and/or recombinant PAV genomes.
Cross-neutralization studies have indicated the existence of at least five serotypes of PAV. Derbyshire et al. (1975) J. Comp. Pathol. 85:437-443; and Hirahara et al. (1990) Jpn. J. Vet. Sci. 52:407-409. Previous studies of the PAV genome have included the determination of restriction maps for PAV Type 3 (PAV-3) and cloning of restriction fragments representing the complete genome of PAV-3. Reddy et al. (1993) Intervirology 36:161-168. In addition, restriction maps for PAV-1 and PAV-2 have been determined. Reddy et al. (1995b) Arch. Virol. 140:195-200.
Nucleotide sequences have been determined for segments of the genome of various PAV serotypes. Sequences of the E3, pVIII and fiber genes of PAV-3 were determined by Reddy et al. (1995a) Virus Res. 36:97-106. The E3, pVIII and fiber genes of PAV-1 and PAV-2 were sequenced by Reddy et al. (1996) Virus Res. 43:99-109; while the PAV-4 E3, pVIII and fiber gene sequences were determined by Kleiboeker (1994) Virus Res. 31:17-25. The PAV-4 fiber gene sequence was determined by Kleiboeker (1995b) Virus Res. 39:299-309. Inverted terminal repeat (ITR) sequences for all five PAV serotypes (PAV-1 through PAV-5) were determined by Reddy et al. (1995c) Virology 212:237-239. The PAV-3 penton sequence was determined by McCoy et al. (1996a) Arch. Virol. 141:1367-1375. The nucleotide sequence of the E1 region of PAV-4 was determined by Kleiboeker (1995a) Virus Res. 36:259-268. The sequence of the protease (23K) gene of PAV-3 was determined by McCoy et al. (1996b) DNA Seq. 6:251-254. The unpublished sequence of the PAV-3 hexon gene (and the 14 N-terminal codons of the 23K protease gene) has been deposited in the GenBank database under accession No. U34592. The unpublished sequence of the PAV-3 100K gene has been deposited in the GenBank database under accession No. U82628. The sequence of the PAV-3 E4 region has been determined by Reddy et al. (1997) Virus Genes 15:87-90.
Adenoviruses have proven to be effective vectors for the delivery and expression of foreign genes in a number of specific applications, and have a number of advantages as potential gene transfer and vaccine vectors. See Gerard et al (1993) Trends Cardiovasc. Med 3:171-177; Imler et al. (1995) Hum. Gene Ther. 6:711-721. The ability of these vectors to mediate the efficient expression of candidate therapeutic or vaccine genes in a variety of cell types, including post mitotic cells, is considered an advantage over other gene transfer vectors. Adenoviral vectors are divided into helper-independent and helper-dependent groups based on the region of the adenoviral genome used for the insertion of transgenes. Helper-dependent vectors are usually made by deletion of E1 sequences and substitution of foreign DNA, and are produced in complementing human cell lines that constitutively express E1 proteins. Graham et al. (1977) J. Gen. Virol. 36:59-74; Fallaux et al. (1996) Hum. Gene Ther. 7:215-222; Fallaux et al. (1998) Hum. Gene Ther. 9:1909-1917. However, porcine adenoviruses do not replicate in human cell lines; hence these lines are unsuitable for the propagation of E1-deleted PAV vectors.
Though E1-deleted viruses do not replicate in cells that do not express E1 proteins, the viruses can express foreign proteins in these cells, provided the genes are placed under the control of a constitutive promoter. Xiang et al. (1996) Virology 219:220-227. Vaccination of animals with adenovirus recombinants containing inserts in the E1 region induced a systemic immune response and provided protection against subsequent challenge. Imler et al (1995) Hum. Gene Ther. 6:711-721; Imler et al. (1996) Gene Therap 3:75-84.. This type of expression vector provides a significant safety profile to the vaccine as it eliminates the potential for dissemination of the vector within the vaccinee and therefore, the spread of the vector to nonvaccinated contacts or to the general environment. However, the currently used human adenovirus (HAV) based vectors are endemic in most populations, which provides an opportunity for recombination between the helper-dependent viral vectors and wild type viruses. To circumvent some of the problems associated with the use of human adenoviruses, non human adenoviruses have been explored as possible expression vectors. All vectors developed to date, except one (Klonjkowski et al (1997) Hum. Gene Ther. 8:2103-2115), contain an intact E1 region. Use of such vectors for gene therapy in humans and vaccination in animals is unsafe because they have the ability to replicate in normal cells, and they retain the oncogenic potential of the E1 region.
Recombinant PAV genomes containing heterologous nucleotide sequences have not yet been described. Similarly, sites where insertion of heterologous sequence would not interfere with the ability of a PAV vector to stimulate an immune response against a determinant encoded by an inserted sequence have not been identified. Consequently, the development of effective recombinant PAV vectors for use in immunization, expression systems and gene therapy, awaits resolution of these issues. Similarly, there is a need for improved adenoviral vectors lacking E1 replication and oncogenic functions, for expression of transgenes in mammalian cells.
The present invention provides the complete nucleotide sequence of the porcine adenovirus type 3 (PAV-3) genome. Nucleic acid sequences that are substantially homologous to those comprising a PAV genome are also encompassed by the invention. Substantially homologous sequences include those capable of duplex and/or triplex formation with a nucleic acid comprising all or part of a PAV genome (or with its complement). As is known to those of skill in the art, duplex formation is influcenced by hybridization conditions, particularly hybridization stringency. Factors affecting hybridization stringency are well-known to those of skill in the art. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual; Hames et al. 1985) Nucleic Acid Hybridisation: A Practical Approach, IRL Press Ltd., Oxford Accordingly, it is within the skill of the art to identify a sequence that is substantially homologous to a sequence from a PAV genome.
In addition, novel porcine adenovirus (PAV) expression vector systems comprising PAV genome sequences are disclosed herein. The PAV-3 sequence includes regions into which heterologous sequences can be inserted including, but not limited to, the E1, E3 and E4 regions, and the region between E4 and the right end of the genome. The invention also provides non-essential regions which can be deleted to increase the capacity of a PAV vector for inserted heterologous sequences. These include, but are not limited to, the E3 and E4 regions, and the region between E4 and the right end of the genome. Essential regions, such as E1, can also be deleted, if virus bearing such deletions are propagated in helper cell lines supplying the deleted essential function. Thus, PAV genome sequences can be replaced by one or more foreign genes to generate recombinant PAV vectors expressing heterologous antigenic polypeptides (or antigenic fragments thereof) for the purposes of producing live recombinant virus, subunit vaccines, nucleic acid immunization, or other types of therapy. Multiple heterologous sequences can be inserted into the same, or different, locations in the genome, limited only by the capacity of the virus to accept heterologous sequences. This capacity can be expanded by deletion of viral sequences.
In addition, the invention provides PAV transcriptional and translational regulatory sequences which can be used for expression of heterologous genes that have been inserted into the vectors of the invention. Furthermore, the novel sequences of the present invention can be used for diagnostic purposes, to determine the presence of PAV antigens and/or PAV nucleic acids in a subject or biological sample.
In additional embodiments, the invention provides compositions providing immunity to PAV infection, through expression of antigenic PAV polypeptides. The invention also provides vectors comprising PAV genome sequences, including sequences encoding various PAV genes as well as PAV regulatory sequences, which are useful for controlling the expression of heterologous genes inserted into PAV vectors.
The invention provides defective recombinant PAV vectors that are deleted in their E1 region, as well as helper cell lines providing E1 function, in which such defective vectors can be propagated. Because these defective vectors replicate inefficiently in cells other than the helper cells, they are less likely to stimulate an immune response in a mammalian host. This makes them particularly suitable for use as vaccine vectors. In addition, since the amount of nucleic acid that can be packaged into an adenovirus virion is limited, deletion of the E1 region expands the capacity of these defective vectors, enabling them to accept larger inserts of heterologous sequence. Additional deletions in other regions of the genome can be used to expand the capacity of these defective vectors still further.
The invention further provides methods for obtaining recombinant PAV vectors. In a preferred embodiment, heterologous nucleotide sequences are introduced, through recombinant DNA techniques, into a bacterial plasmid comprising a defined portion of the PAV genome. The recombinant plasmid, containing heterologous sequences flanked by PAV sequences, is introduced into a host cell in combination with a full-length PAV genome or a plasmid containing a full-length or nearly full-length PAV genome. Within the host cell, recombination between the plasmid and the PAV genome generates a recombinant PAV genome. Alternatively, recombinant PAV genomes can be constructed in vitro, using standard techniques in molecular biology and biotechnology.
The invention also provides methods for preparing live recombinant virus and subunit vaccines for inducing protective immune responses to an infectious organism in a mammalian subject. Protective immune responses include humoral (antibody) responses, cell-mediated responses, mucosal responses, or any combination of these. The methods involve insertion, into the porcine adenovirus genome, of heterologous nucleotide sequences encoding one or more protective antigenic determinants of a pathogen. The heterologous sequences are inserted in such a way as to come under the regulatory control of a PAV promoter, or the heterologous sequences are inserted in operative linkage to a eukaryotic transcriptional regulatory sequence. Translation of transcribed heterologous sequences can be controlled by PAV translational regulatory elements, or the heterologous sequence can include non-PAV sequences which regulate its translation.
In another aspect, the invention includes the use of recombinant porcine adenoviruses and recombinant PAV vectors for the expression of a nucleotide or amino acid sequence of interest in a cell system, such as, for example, production of antigen to be used in the preparation of antibodies, or production of antisense RNA.
The invention also includes an expression system comprising a porcine adenovirus expression vector wherein heterologous nucleotide sequences are inserted. The inserted heterologous sequences can comprise one or more regulatory elements for transcription and/or translation, or can be inserted so as to come under the control of PAV regulatory elements. Inserted regulatory elements can be those that are normally associated with the heterologous sequence, or a heterologous sequence can be juxtaposed to and placed in operative linkage with a regulatory element with which it is not normally associated, using standard recombinant DNA techniques. Heterologous sequences can be inserted into a full-length PAV genome, or into a PAV genome which has been deleted in one or more regions. A deletion in the PAV genome can be made to provide a site for insertion of a heterologous sequence, or simply to increase the capacity of the PAV vector to accommodate heterologous sequences inserted at another location.
The invention also provides recombinant PAV polypeptides including, but not limited to, those encoded by the following genes: E1A, E1B, E4, pIX, DBP, pTP, pol, IVa2, 52K, IIIA, pIII, pVII, pV, pX, pVI, and 33K. Such recombinant PAV polypeptides are produced in any eukaryotic expression vector known in the art, into which is inserted a PAV nucleotide sequence according to the invention. Also provided are methods and compositions for recombinant production of heterologous polypeptides and RNAs in a PAV vector. Expression of heterologous polypeptides and RNAs in a PAV vector can be regulated by endogenous PAV regulatory sequences, or by non-PAV sequences. Non-PAV regulatory sequences can be those which normally regulate the heterologous sequence, or they can be sequences that are not normally associated with the heterologous sequence in a regulatory capacity.
Thus, in one embodiment, the invention includes an expression system in which one or more regions of the PAV genome are deleted and replaced with heterologous sequences. In another embodiment, the invention includes a PAV expression system in which heterologous sequences are introduced into the PAV genome without the removal of any PAV sequences. Intergenic regions of the PAV genome comprising regulatory sequences are useful in the practice of the invention for controlling the expression of homologous and heterologous sequences.
The invention also includes recombinant vector systems comprising two or more nucleic acid molecules. In one embodiment, the vector system comprises two plasmids, the first containing a full-length or nearly full-length PAV genome and the second containing a segment of the PAV genome, such as the left end (including the E1 region) or the right end (including the E3 and/or E4 regions). Introduction of heterologous nucleotide sequences into the second plasmid, followed by co-transfection of both plasmids into a suitable host cell, will allow homologous recombination between the two plasmids to generate a viral genome containing inserted heterologous sequences. In another embodiment, the vector system comprises a full-length or nearly full-length PAV genome and a plasmid containing a segment of the PAV genome. Insertion of heterologous sequences into the plasmid, followed by co-transfection and homologous recombination, will generate recombinant PAV genomes as above.
Additional aspects of the invention provide a recombinant PAV comprising a heterologous sequence wherein the heterologous sequence encodes an antigenic determinant of a disease-causing organism; and a recombinant PAV comprising a heterologous sequence wherein the heterologous sequence encodes a foreign gene or fragment thereof. In further embodiments, the invention provides pharmaceutical compositions comprising recombinant PAV for producing an immune response in a mammalian host, the recombinant PAV comprising a heterologous nucleotide sequence encoding a protective determinant of a pathogenic organism. The heterologous sequence is expressed in quantities sufficient for induction of a protective immune response, either through operative linkage to one or more non-PAV regulatory sequences, or through control by endogenous PAV regulatory sequences. The protective immune response can be humoral, cell-mediated and/or mucosal.
The recombinant PAV vectors of the invention will also allow the expression of various therapeutic polypeptides in a wide range of mammalian hosts and are thus useful in the practices of nucleic acid immunization and gene therapy. Exemplary hosts include, but are not limited to, human, equine, bovine, porcine, ovine, caprine, avian, and murine. Those of skill in the art are aware of various therapeutic polypeptides which can be usefully expressed in mammalian hosts. Such therapeutic polypeptides include, but are not limited to, coagulation factors, growth hormones, cytokines, lymphokines, tumor-suppressing polypeptides, cell receptors, ligands for cell receptors, protease inhibitors, antibodies, toxins, immunotoxins, dystrophins, cystic fibrosis transmembrane conductance regulator (CFTR) and immunogenic polypeptides.
The invention also provides diagnostic methods and compositions for the detection of PAV nucleic acids and proteins in a cell or biological sample. The PAV nucleotide sequences disclosed herein can be used as hybridization probes to detect PAV nucleic acids. In addition, the PAV nucleotide sequences disclosed herein encode PAV polypeptides, which can be used for the production of antibodies reactive with various PAV antigens. Such antibodies can be used to detect PAV antigens by immunoassay. Alternatively, PAV polypeptides themselves can be used in competitive immunoassays to detect the presence of PAV antigens in a cell or biological sample. PAV polypeptides can be produced by the PAV vectors of the invention, or can be produced in any mammalian expression vector known in the art.